Synthesis of 7α-Methoxy-7-(4-phenyl-1H-1,2,3-triazol-1-yl)acetamino-3′-arylthio-cephalosporic Acid Derivatives from 7-Aminocephalosporic Acid

The aim of this project was to develop a synthetic protocol for the preparation of a cephamycin scaffold that would readily allow the synthesis of its analogues with variations at the C-7 amino group and the C-3′ position. We also aimed to develop a method that avoided the use of toxic and potentially explosive diphenyldiazomethane. These aims were achieved via the synthesis of the novel α-bromo acetamide 18 which allowed functionalization at the α-bromo acetamide position by azide and then the introduction of a 4-phenyl-1H-1,2,3-triazol-1-yl moiety via a Cu(I)-catalysed azide–alkyne cycloaddition reaction with phenylacetylene. Palladium-catalyzed arylthioallylation reactions then allowed the introduction of 3′-arylthiol substituents. We also report for the first time the synthesis of the 4-methoxybenzyl ester of (6R,7S)-3-[(acetyloxy)methyl]-7-amino-7-methoxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid and the use of diphenyl trichloroacetimidate, instead of diphenyldiazomethane, and 4-methoxybenzyl trichloroacetimidate to prepare related 4-methoxybenzyl esters. The chemistry described, and several of the synthetic intermediates reported here, are potentially valuable methods and scaffolds, respectively, for further development of β-lactam antibiotics.


Introduction
The penicillins, cephalosprins, and cephamycins are important classes of clinically used β-lactam antibiotics [1].Well-known representatives of each of these groups are penicillin N 1, cephalosporium C 2, and cephamycin C 3, respectively.The latter two compounds are biosynthetically connected to penicillin N 1 by ring-expansion and, in the case of cephamycin C 3, enzymatic introduction of a 7-α-methoxy substituent (Scheme 1) [1].Examples of important cephamycin antibiotics are cefotetan 4, cefoxitin 5, and cefmetazole 6 (Figure 1) which are ascribed as second-generation cephalosporins with broad-spectrum in vitro antibacterial activity.Additionally, these compounds have anti-anaerobic activities making these valuable agents against intraabdominal infections [2].Furthermore, their 7αmethoxy substituent decreases their vulnerabilities to β-lactamases [1], which potentially increases their antibacterial efficacies.
In this paper we report our study on the synthesis of the diphenylmethyl ester 7 from 7-ACA 9 that avoids the use of toxic and potentially explosive diphenyldiazomethane [6] and the synthesis of the 4-methoxybenzyl (PMB) ester 8 from 7-ACA 9 and demonstrate here their application to the synthesis of derivatives of the type 10.While compound 8 has been prepared previously, its synthesis is only described in the patent literature where only racemic 8 was prepared and very little characterization data for 8 or its precursors were described [7][8][9].
The main challenge in the synthesis of 7 and 8 from 9 was the introduction of the 7αmethoxy group.This has been achieved from the diphenylmethyl ester of 7-ACA 9 via treatment with HNO2, to give the 7-diazoderivative, followed by treatment with potentially explosive bromo azide to give a diastereomeric mixture of bromo azides.This mixture was then treated with methanol/AgBF4 to give the corresponding 7-azido-7methoxy derivative and then hydrogenation gave 7 [10].Lunn and Mason prepared 7, via protection of the 7-amino group of 7-ACA 9 as a carbamate derivative and then esterification with diphenyldiazomethane [11].The method of Koppel [12] was then employed via treatment of this diprotected compound using an excess amount of base (3.5 equiv.lithium methoxide) in tetrahydrofuran solvent and then tert-butyl hypochlorite at a low temperature (−80 °C) to generate a C-7 imine intermediate that was captured using the excess methoxide resulting in a C-7-aminocarbamate-C-7α-methoxy derivative.This then required carbamate deprotection via hydrogenolysis to give 7 as an unstable compound.An alternative procedure, that does not require potentially explosive reagents or intermediates or the use of strong base at low temperatures, was reported by Yanagisawa et al. [13,14].This method involves oxidation of the Schiff base formed from the reaction of the diphenylmethyl ester of 7-ACA 9 with 3,5-di-tert-butyl-4hydroxybenzaldehyde with lead dioxide and then treatment of the resulting C-7 imine with methanol.The imine of the resulting methanol adduct was then cleaved upon exposure to Girard-T reagent ((carboxymethyl)trimethylammonium chloride hydrazide) to give 7. Yoshida later reported that lead dioxide could be replaced with 2,3-dichloro-5,6dicyano-1,4-benzoquinone (DDQ) [15].We chose to employ the method of Yoshida using DDQ as the oxidant.

Results and Discussion
We initially investigated the synthesis of diphenylmethyl ester 11 from 7-ACA 9.As indicated in (Scheme 3a) this has been prepared in 65% yield from the reaction of 9 with potentially hazardous diphenyldiazomethane (Scheme 3a) [16].In a model study, we found that the known diphenylmethyl ester 13 [17] could be obtained in 88% yield from the reaction of acid 12 [18] and diphenyl trichloroacetimidate [19,20] in dichloromethane The main challenge in the synthesis of 7 and 8 from 9 was the introduction of the 7α-methoxy group.This has been achieved from the diphenylmethyl ester of 7-ACA 9 via treatment with HNO 2 , to give the 7-diazoderivative, followed by treatment with potentially explosive bromo azide to give a diastereomeric mixture of bromo azides.This mixture was then treated with methanol/AgBF 4 to give the corresponding 7-azido-7-methoxy derivative and then hydrogenation gave 7 [10].Lunn and Mason prepared 7, via protection of the 7-amino group of 7-ACA 9 as a carbamate derivative and then esterification with diphenyldiazomethane [11].The method of Koppel [12] was then employed via treatment of this diprotected compound using an excess amount of base (3.5 equiv.lithium methoxide) in tetrahydrofuran solvent and then tert-butyl hypochlorite at a low temperature (−80 • C) to generate a C-7 imine intermediate that was captured using the excess methoxide resulting in a C-7-aminocarbamate-C-7α-methoxy derivative.This then required carbamate deprotection via hydrogenolysis to give 7 as an unstable compound.An alternative procedure, that does not require potentially explosive reagents or intermediates or the use of strong base at low temperatures, was reported by Yanagisawa et al. [13,14].This method involves oxidation of the Schiff base formed from the reaction of the diphenylmethyl ester of 7-ACA 9 with 3,5-di-tert-butyl-4-hydroxybenzaldehyde with lead dioxide and then treatment of the resulting C-7 imine with methanol.The imine of the resulting methanol adduct was then cleaved upon exposure to Girard-T reagent ((carboxymethyl)trimethylammonium chloride hydrazide) to give 7. Yoshida later reported that lead dioxide could be replaced with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) [15].We chose to employ the method of Yoshida using DDQ as the oxidant.

Results and Discussion
We initially investigated the synthesis of diphenylmethyl ester 11 from 7-ACA 9.As indicated in (Scheme 3a) this has been prepared in 65% yield from the reaction of 9 with potentially hazardous diphenyldiazomethane (Scheme 3a) [16].In a model study, we found that the known diphenylmethyl ester 13 [17] could be obtained in 88% yield from the reaction of acid 12 [18] and diphenyl trichloroacetimidate [19,20] in dichloromethane (CH 2 Cl 2 ) solvent after 1 h at room temperature (rt) (Scheme 3b).However, our attempts to prepare 11 directly from 7-ACA 9 under similar reaction conditions were not successful due to the poor solubility of 9 in CH 2 Cl 2 .To prepare a more soluble substrate, a suspension of 9 in CH 2 Cl 2 was treated first with N,O-bis(trimethylsilyl)acetamide (BSA) [21] to give a solution of the corresponding trimethylsilyl ester in situ followed by the addition of diphenyl trichloroacetimidate.However, only trace amounts of the desired product ( 11) could be detected using electron impact mass spectrometric (ESIMS) analysis (Scheme 3c).
(CH2Cl2) solvent after 1 h at room temperature (rt) (Scheme 3b).However, our attempts to prepare 11 directly from 7-ACA 9 under similar reaction conditions were not successful due to the poor solubility of 9 in CH2Cl2.To prepare a more soluble substrate, a suspension of 9 in CH2Cl2 was treated first with N,O-bis(trimethylsilyl)acetamide (BSA) [21] to give a solution of the corresponding trimethylsilyl ester in situ followed by the addition of diphenyl trichloroacetimidate.However, only trace amounts of the desired product ( 11) could be detected using electron impact mass spectrometric (ESIMS) analysis (Scheme 3c A more successful pathway was realized via the formation of the Schiff base 14 from the reaction of 7-ACA 9 first with BSA in dimethylacetamide (DMA) as a solvent and then treatment with 3,5-di-tert-butyl-4-hydroxybenzaldehyde (Scheme 4).This reaction mixture was then treated with diphenyl trichloroacetimidate to give the known diphenylmethyl ester 15a [22].Both compounds 14 and 15a proved to be unstable to purification via column chromatography; however, the formation of imine 14 was evident in 1 H NMR analysis of the crude reaction mixture ( 1 H NMR (500 MHz,CD3SOCD3) in part, 8.44 (s, 1H, CH=NAr), 7.57 (s, 2H, ArCH), and 1.39 (s, 18H, 2 × C(CH3)3) ppm) and low resolution mass spectrometric (LRMS) analysis (ESI +ve) which showed an ion peak at m/z 489 ([M + H] + , 58%).While the formation of compound 15a was evident from 1 H NMR analysis of the crude reaction mixture ( 1 H NMR (500 MHz, CD3OD) in part, 8.45 (s, 1H, CH=NAr) and 6.94 (s, 1H, CHPh2) ppm) it also contained unreacted 3,5-di-tert-butyl-4hydroxybenzaldehyde and DMA.Oxidation of this mixture with DDQ in methanol solvent gave the known 7α-methoxylated imine derivative 16a [13] in an optimized 22% overall yield for the three steps from 9. Key to this optimized yield was performing the purification of 16a using column chromatography below ambient temperature to prevent imine hydrolysis (see Experimental section for details).Treatment of 16a with the Girard-T reagent gave a mixture of the known compound 7 [23] and the di-tert-butyl-4hydroxybenzaldehyde imine of Girard's reagent.Attempts to purify 7 were unsuccessful A more successful pathway was realized via the formation of the Schiff base 14 from the reaction of 7-ACA 9 first with BSA in dimethylacetamide (DMA) as a solvent and then treatment with 3,5-di-tert-butyl-4-hydroxybenzaldehyde (Scheme 4).This reaction mixture was then treated with diphenyl trichloroacetimidate to give the known diphenylmethyl ester 15a [22].Both compounds 14 and 15a proved to be unstable to purification via column chromatography; however, the formation of imine 14 was evident in 1 H NMR analysis of the crude reaction mixture ( 1 H NMR (500 MHz,CD 3 SOCD 3 ) in part, 8.44 (s, 1H, CH=NAr), 7.57 (s, 2H, ArCH), and 1.39 (s, 18H, 2 × C(CH 3 ) 3 ) ppm) and low resolution mass spectrometric (LRMS) analysis (ESI +ve) which showed an ion peak at m/z 489 ([M + H] + , 58%).While the formation of compound 15a was evident from 1 H NMR analysis of the crude reaction mixture ( 1 H NMR (500 MHz, CD 3 OD) in part, 8.45 (s, 1H, CH=NAr) and 6.94 (s, 1H, CHPh 2 ) ppm) it also contained unreacted 3,5-di-tert-butyl-4-hydroxybenzaldehyde and DMA.Oxidation of this mixture with DDQ in methanol solvent gave the known 7α-methoxylated imine derivative 16a [13] in an optimized 22% overall yield for the three steps from 9. Key to this optimized yield was performing the purification of 16a using column chromatography below ambient temperature to prevent imine hydrolysis (see Experimental section for details).Treatment of 16a with the Girard-T reagent gave a mixture of the known compound 7 [23] and the di-tert-butyl-4hydroxybenzaldehyde imine of Girard's reagent.Attempts to purify 7 were unsuccessful due to product instability; thus, the mixture was treated with bromoacetyl bromide and pyridine to give the more stable and novel α-bromo acetamide 17a in 42% yield from 16a (Scheme 4) after purification via column chromatography.In our studies of related acylation reactions, we found that low temperatures and short reaction times were essential to prevent isomerization of the C-2 double bond to the ∆-3 isomer [24].This procedure was then applied to the synthesis of the PMB ester 8, using commercially available 4methoxyphenyl trichloroacetimidate rather than diphenyl trichloroacetimidate, and then its corresponding and novel α-bromoacetamide 17b (Scheme 4).The ester 8 was able to be purified via column chromatography at ambient temperature; however, decomposition of this compound was observed during its characterization process, as evidenced by the color change of the sample (green to orange) and 1 H NMR analysis.It was, therefore, used directly for the subsequent reaction without further purification.This latter synthetic protocol proved more convenient due to the commercial availability of 4-methoxyphenyl trichloroacetimidate and overall yields and gave a more stable intermediate-PMB ester 8.
pyridine to give the more stable and novel α-bromo acetamide 17a in 42% yield from 16a (Scheme 4) after purification via column chromatography.In our studies of related acylation reactions, we found that low temperatures and short reaction times were essential to prevent isomerization of the C-2 double bond to the Δ-3 isomer [24].This procedure was then applied to the synthesis of the PMB ester 8, using commercially available 4-methoxyphenyl trichloroacetimidate rather than diphenyl trichloroacetimidate, and then its corresponding and novel α-bromoacetamide 17b (Scheme 4).The ester 8 was able to be purified via column chromatography at ambient temperature; however, decomposition of this compound was observed during its characterization process, as evidenced by the color change of the sample (green to orange) and 1 H NMR analysis.It was, therefore, used directly for the subsequent reaction without further purification.This latter synthetic protocol proved more convenient due to the commercial availability of 4-methoxyphenyl trichloroacetimidate and overall yields and gave a more stable intermediate-PMB ester 8. Compounds 17a and 17b are attractive intermediates for the synthesis of analogues related to the general structure 10 via the introduction of other substituents at the αacetamide and the C-3 positions.To explore this potential, we have converted 17a and 17b to the carboxylic acid 18 via treatment with trifluoroacetic acid (TFA) (Scheme 5).Treatment of this acid with sodium azide in dimethyl formamide (DMF) at −5 • C gave the azide 19 in 81% yield.We found that higher reaction temperatures led to mixtures of 19 and its double-bonded shifted isomer (∆-3 isomer of 19).The azide 19 was subjected to a Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC) reaction [25] with phenylacetylene at 30 • C for 24 h, which provided the triazole 20 in 66% yield (Scheme 5).While the triazole 20 is a new compound, the corresponding 1H-tetrazole derivative has been reported in the patent literature [26].
azide 19 in 81% yield.We found that higher reaction temperatures led to mixtures of 19 and its double-bonded shifted isomer (Δ-3 isomer of 19).The azide 19 was subjected to a Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC) reaction [25] with phenylacetylene at 30 °C for 24 h, which provided the triazole 20 in 66% yield (Scheme 5).While the triazole 20 is a new compound, the corresponding 1H-tetrazole derivative has been reported in the patent literature [26].We next focused on functionalization of the C-3′ position via displacement of the Oacetyl group with an arylthiol moiety using the palladium-catalyzed thioallylation method reported by Breinbauer et al. using 2 mol% bis(dibenzylideneacetone)palladium( 0 as a ligand and acetonitrile as a solvent [27].This method had been successfully applied by Breinbauer et al. to the cephalosporin antibiotic cefalotin, which, unlike 20, bears a C-7 2thienylacetamido substituent and lacks the 7α-methoxy group.Pd-catalyzed reactions of cefalotin with 4-methylthiophenol and 4-fluorothiophenol gave the corresponding C-3′ arylthiol derivatives in yields of 41% and 58%, respectively [27].
We initially studied the thioallylation reaction of 20 under similar reaction conditions, except using 1 mol% (2 mol% Pd) of tris(dibenzylideneacetone)dipalladium(0)-chloroform (Pd2(dba)3.CHCl3) as the palladium source (Table 1).However, after stirring the reaction under an argon atmosphere for 5 d We next focused on functionalization of the C-3 position via displacement of the Oacetyl group with an arylthiol moiety using the palladium-catalyzed thioallylation method reported by Breinbauer et al. using 2 mol% bis(dibenzylideneacetone)palladium( 0 as a ligand and acetonitrile as a solvent [27].This method had been successfully applied by Breinbauer et al. to the cephalosporin antibiotic cefalotin, which, unlike 20, bears a C-7 2-thienylacetamido substituent and lacks the 7α-methoxy group.Pd-catalyzed reactions of cefalotin with 4-methylthiophenol and 4fluorothiophenol gave the corresponding C-3 arylthiol derivatives in yields of 41% and 58%, respectively [27]. We initially studied the thioallylation reaction of 20 under similar reaction conditions, except using 1 mol% (2 mol% Pd) of tris(dibenzylideneacetone)dipalladium(0)-chloroform (Pd 2 (dba) 3 .CHCl 3 ) as the palladium source (Table 1).However, after stirring the reaction under an argon atmosphere for 5 d at 35 • C, only unreacted 20 was evident from 1 H NMR and MS analysis of the crude reaction mixture (Table 1, Entry 1).Similar results were obtained using 2 mol% triphenyl phosphite (P(OPh) 3 ) or 1,1 -ferrocenediyl-bis(diphenylphosphine) (dppf) as the ligand (Table 1, Entries 2 and 3, respectively).We discovered, however, that performing the reaction using conditions of Entry 1 under sonication resulted in 70% conversion to the desired thiol derivative 21a after 12 h (Table 1, Entry 4).Increasing the Pd and ligand loadings to 20 mol% and the equivalents of the thiol to 2.0 equiv., under sonication conditions, resulted in full conversion of 20 to 21a (Table 1, Entry 5).After a standard work-up procedure, the crude product was purified using semi-preparative RP-HPLC to give 21a in 23% yield and in 99% purity via analytical HPLC analysis (Scheme 4).Thiol derivatives 21b-21h were then prepared under similar reaction conditions and purified using RP-HPLC with the yields shown in Scheme 4. In each case the analytical purities of these thiolated products were >99%, except for 21b which had a purity of 98.7%.The 4-nitrophenylthio and the 4-chlorophenylthio derivatives 21i and 21j, respectively, could not be obtained using this synthetic protocol (Scheme 5).
Table 1.Attempts toward the synthesis of thiol 21a from triazole 20.

General Statement
Unless stated otherwise, all solvents and chemicals were laboratory-or reagent-grade and were purchased from commercial sources.Anhydrous solvents were obtained from a solvent dispenser under nitrogen and stored over 4 Å molecular sieves.All chemicals were used as received.All reactions were conducted under normal atmosphere with air or nitrogen.Cold reaction temperatures were obtained by using an ice bath (0 • C) or ice/salt bath (-20 • C).Heating of reactions was performed using a paraffin oil bath.Small quantities of liquid reagents were measured and added to reactions via syringe or autopipette.All filtrations were gravity filtrations through cotton wool or filter paper in a glass funnel.Solvent removal via concentration was performed on a rotary evaporator under reduced pressure.All synthesized compounds were dried under high vacuum (~1 mbar) before determination of chemical yields and spectroscopic characterization.All solvent mixtures are expressed in terms of volume ratio (i.e., v/v).Flash chromatography was performed using Chem Supply silica gel 60 230-400 mesh.Thin layer chromatography (TLC) was performed on Merck aluminum-backed SiO 2 gel plates (F254 grade-0.20 mm thickness).A reversed-phase (RP) C18 (Synergi™ 4 µm Fusion-RP 80 Å (Elkridge, MD, USA), LC Column 150 × 4.6 mm) column was used with a MeCN/H 2 O (0:100-100:0) gradient mobile phase containing 0.01% TFA at a flow rate of 1.0 mL/min for the analysis.Compounds were detected using UV-vis at 279 or 254 nm, depending on their highest absorption.Visualization was achieved using UV light and cerium ammonium molybdate stain.All known compounds are marked with a reference after the compound title and all other compounds without a reference are novel.

Characterization and Analysis
All novel compounds were subjected to full spectroscopic characterization and assignment based on 2-D NMR experiments. 1 H NMR spectra were recorded on a Bruker Avance400 (400 MHz) and Bruker Avance500 (500 MHz) (Billerica, MA, USA).Chemical shifts are reported in ppm and were measured relative to the internal standard.Samples were dissolved in CDCl 3 (with TMS as the internal standard-0.00ppm) and CD 3 OD (solvent resonance as internal standard-3.31ppm).The 1 H NMR data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, ABq = AB quartet, m = multiplet, br = broad), coupling constants (Hz), integration, and assignment. 13C NMR spectra were recorded on a Bruker Avance 400 (100 Hz) and Bruker Avance 500 (125 MHz) NMR spectrometer with complete 1 H decoupling.Chemical shifts are reported in ppm and were measured relative to the internal standard.Samples were dissolved in CDCl 3 (solvent resonance as internal standard-77.16ppm) and CD 3 OD (solvent resonance as internal standard-49.0ppm). 1 H and 13 C NMR signal assignments were confirmed via analysis of 2-D NMR experiments: gCOSY, gHSQC, and gHMBC.The abbreviations section defines all NMR experiment acronyms.All NMR spectra were processed, analyzed, and prepared with MestReNova (version 12.0) NMR software.Low resolution mass spectra (LRMS) were obtained via electrospray ionization (ESI) on a Shimadzu LC-2010 mass spectrometer (Kyoto, Japan).LRMS data were recorded as the ion mass/charge ratio (m/z) with the corresponding relative abundance as a percentage.High-resolution mass spectrometry (HRMS) was performed on a Waters Quadrupole Time of Flight (QTOF) Xevo spectrometer via ESI and with Leucine-Enkephalin as an internal standard.All mass spectrometry samples were dissolved in high-performance liquid chromatography (HPLC)-grade MeOH.Rotation values (α) are expressed in units of "deg cm 3 g −1 dm −1 " with concentration (c) expressed in units of "g/100 mL".Solid-state infrared spectroscopy was performed on a Bruker Vertex 70 FTIR spectrometer.IR peaks are reported as the wavenumber (ν max in cm -1 ) of the maximum absorption, and the intensities were expressed as s = strong, m = medium, or w = weak.The purity of all tested compounds was determined using analytical HPLC-Waters 1525 binary HPLC pump with a Waters 2487 dual-absorbance detector (column, Synergi Fusion-RP 80Å, 4. To a suspension of 7-ACA 9 (307.4,1.13 mmol) in anhydrous DMA (5 mL) under N 2 at rt, BSA (469.3 µL, 1.92 mmol, 1.7 equiv.) was added and stirred for 30 min until the mixture turned clear.The reaction mixture was then cooled to −20 • C (NaCl/ice), and phenylacetyl chloride (179.2 µL, 1.36 mmol, 1.2 equiv.) was added dropwise.The resulting solution was stirred at −20 • C for 2 h, at which point the reaction was shown to be complete via TLC analysis (TLC (MeOH/CH 2 Cl 2 -2:3): R f = 0.63).The reaction mixture was poured into iced water (20 mL) and extracted using EtOAc (3 × 20 mL).The combined EtOAc layer was washed with water (3 × 20 mL), brine (20 mL), dried over anhydrous MgSO 4 , filtered, and concentrated in vacuo to give a pale-yellow residue.The obtained crude product was then dissolved in a minimum amount of EtOAc, and hexanes were added dropwise to this vigorously stirred solution until precipitation started to occur.The mixture was stirred at rt overnight.The solvent was removed using a syringe and the precipitate was washed with hexanes (5 × 10 mL), then dried in vacuo to afford the titled compound as an off-white powder (258.1 mg, 0.661 mmol, 58%). 1  Benzhydryl 2,2,2-trichloroacetimidate [19,20] To a solution of diphenylmethanol (317.3 mg, 1.72 mmol) in anhydrous CH 2 Cl 2 (2 mL) DBU (25.8 µL, 0.172 mmol, 0.1 equiv.)and CCl 3 CN (1.73 mL, 17.2 mmol, 10 equiv.)were added at rt under an Ar atmosphere.The reaction mixture was stirred at 40 • C overnight, at which point the reaction was shown to be complete via TLC analysis (TLC (3% Et 3 N in toluene): R f = 0.71).Reaction mixture was concentrated in vacuo and purified via flash chromatography over SiO 2 (3% Et 3 N in hexane/EtOAc-80:1) to give the titled compound as a white solid (375.8 mg, 1.14 mmol, 66%). 1 H NMR (500 MHz, CDCl 3 ) δ 8.41 (s, 1H, NH), 7.50-7.23(m, 10H, ArCH), 6.94 (s, 1H, CHPh 2 ); the 1 H NMR spectroscopic data agreed with those previously reported [19,20].

Alternative purification method for scale-up reactions (≥200 mg of 7-ACA 9).
A series of solvents in a EtOAc/hexane system were prepared (using 3.6 mmol crude product as an example: 100 mL-1:19, 200 mL-1:9, 100 mL-3:17, 100 mL-1:4, 100 mL-11:39, 100 mL-6:19, 100 mL-13:37, 100 mL-7:18, and 200 mL-3:7) and chilled at −20 • C overnight.On the following day, an appropriately sized column was packed with a SiO 2 slurry in hexane and chilled at −20 • C until ready to use.Once the crude product was loaded onto the column, the pre-chilled solvents was kept cool on an ice bath and the flow rate was accelerated by using a stream of compressed air to ensure the entire purification process was kept within 30 min. 1  To a solution of 16a (56.4 mg, 0.0824 mmol) in EtOAc (0.5 mL) a solution of Girard-T reagent (27.6 mg, 0.165 mmol, 2.0 equiv.) was added in MeOH (0.6 mL) at rt, and the resulting solution was stirred for 2.5 h until the completion of the reaction was indicated by MS analysis.Upon completion, the reaction mixture was diluted with EtOAc (15 mL) and poured into water (20 mL), which was extracted with additional EtOAc (15 mL × 2).The combined EtOAc layer was washed with brine (20 mL), dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to give the crude product as a dark green gum.Further purification of the crude product resulted in decomposition and it was, therefore, used directly for the subsequent reaction. 1 To a solution of crude product 7 (0.0813 mmol, prepared from 55.6 mg of 16a from the previous reaction in anhydrous CH 2 Cl 2 (1 mL) under N 2 at −20 • C (NaCl/ice) pyridine (20 µL, 0.248 mmol, 3.1 equiv.) was added and the resulting solution was stirred for 2 min.Bromoacetyl bromide (19 µL, 0.216 mmol, 2.7 equiv.) was then added dropwise to the solution.The reaction mixture was stirred at −20 • C for 2 h, at which point the reaction was shown to be complete via TLC analysis (TLC (EtOAc/hexane-2:3): R f = 0.39).The reaction mixture was poured into EtOAc (5 mL); washed sequentially with HCl (1.0 M-5 × 5 mL), saturated NaHCO 3 (5 × 5 mL), and brine (10 mL); dried over anhydrous Na 2 SO 4 ; filtered; and concentrated in vacuo to give a brown residue.The crude product was purified via flash chromatography over SiO 2 (EtOAc/hexane-1:19-9:11) to give the titled compound as a pale-yellow foam (20.3 mg, 0.0344 mmol, 42% from 16a).
[α] To a suspension of crude product 14 (0.757 mmol, prepared from 206.1 mg of 7-ACA 9) in CH 2 Cl 2 (2 mL) under an Ar atmosphere at rt 4-methoxybenzyl 2,2,2-trichloroacetimidate (204.3 µL, 0.984 mmol, 1.3 equiv.) was added, and the resulting mixture was stirred at rt for 48 h.The color of the reaction turned from bright yellow to brown, and the completion of the reaction was indicated using MS analysis.The reaction mixture was filtered and concentrated in vacuo to give the crude product 15 as a sticky brown gum.A portion of this filtrate was used for NMR analysis, which indicated a mixture of the titled compound, DMA, and 3,5-di-tert-butyl-4-hydroxybenzaldehyde.Further purification using flash chromatography resulted in decomposition. 1  The crude compound 15b was suspended in MeOH (5 mL) at −20 • C under an Ar atmosphere.To the suspension DDQ (171.8 mg, 0.757 mmol, 1.0 equiv.) was added, and the reaction mixture was stirred at −20 • C for 45 min.The completion of the reaction was indicated via TLC analysis (TLC (EtOAc/hexane-2:3): R f = 0.39).The reaction mixture was concentrated in vacuo and purified via column chromatography over SiO 2 .Prior to the purification, a series of solvents in a EtOAc/hexane system were prepared (using 3.6 mmol crude product as an example: 100 mL-1:19, 200 mL-1:9, 100 mL-3:17, 100 mL-1:4, 100 mL-11:39, 100 mL-6:19, 100 mL-13:37, 100 mL-7:18, 200 mL-3:7, and 200 mL-7: 20) and chilled at −20 • C overnight.On the following day, an appropriately sized column was packed with a SiO 2 slurry in hexane and chilled at −20 • C until ready to use.Once the crude product was loaded onto the column, the pre-chilled solvents was kept cool on an ice bath and the flow rate was accelerated by using a stream of compressed air to ensure the entire purification process was kept within 30 min.Fractions with R f value of 0.39 (TLC (EtOAc/hexane-2:3)) were combined and concentrated in vacuo to give the title compound as a hydroscopic, pale-yellow foam (204.1 mg, 0.320 mmol, 42% from 7-ACA).
[α] To a solution of 16b (333.5 mg, 0.487 mmol) in EtOAc (2.4 mL) a solution of Girard-T reagent (163.3 mg, 0.974 mmol, 2.0 equiv.) in MeOH (2.9 mL) at rt was added, and the resulting solution was stirred for 3.5 h until the completion of the reaction was indicated via TLC analysis (TLC (EtOAc/hexane-1:1): R f = 0.24).Upon completion, the reaction mixture was diluted with EtOAc (50 mL) and poured into water (50 mL), which was extracted using additional EtOAc (50 mL × 2).The combined EtOAc layer was washed with brine (70 mL), dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to give the crude product as a dark green gum.A portion of this crude product (0.216 mmol) was purified via flash chromatography over SiO 2 (EtOAc/hexane-1:10-2:3) to give the titled compound as a green gum (15.4 mg, 0.0365 mmol, 17%).
Method 2-prepared from compound 17b.To a solution of compound 17b (67.5 mg, 0.124 mmol) in anhydrous CH 2 Cl 2 (1 mL) at 0 • C TFA was slowly added (247 µL, 3.23 mmol, 26 equiv.).The resulting dark-pink solution was stirred at 0 • C for 30 min.Work-up and precipitation as in Method 1 gave the titled compound as a yellow gum (47.1 mg, 0.111 mmol, 90%  To a solution of compound 18 (36.7 mg, 0.0867 mmol) in DMF (0.3 mL) at −15 • C sodium azide (28.2 mg, 0.434 mmol, 5.0 equiv.) was added, and the solution was stirred for 24 h, at which point the reaction was shown to be complete via MS analysis.The reaction mixture was diluted with distilled H 2 O (20 mL), to which EtOAc (20 mL) was added.The resulting mixture was stirred vigorously, and the aqueous layer was acidified with conc.HCl to pH < 1.The two layers were separated, and the aqueous layer was extracted with EtOAc (3 × 20 mL).The combined organic layer was washed sequentially with distilled H 2 O (3 × 20 mL) and brine (40 mL), dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to give a sticky gum.This crude product was dissolved in a minimum amount of EtOAc, and hexane was added dropwise to the vigorously stirred solution until the precipitation started to occur.The mixture was stirred at rt overnight.The solvent was removed using a syringe and the precipitate was washed with hexanes (5 × 10 mL), then dried in vacuo to afford the titled compound as a pale-yellow gum (27.1 mg, 0.0703 mmol, 81%).For NMR assignments of compounds 20 and 21a-h, the following numbering system has been used.To this stirred mixture, phenylacetylene (23.3 μL, 0.211 mmol, 3.0 eq.) was and the reaction mixture was stirred at 30 ℃ for 24 h.The reaction mixture with EtOAc (20 mL), washed with saturated aqueous NH4Cl solution (20 mL anhydrous MgSO4, filtered, and concentrated in vacuo.The obtained resid dissolved in a minimum amount of EtOAc, and to this vigorously stirred solu were added dropwise until precipitation started to occur.The mixture was overnight.The solvent was removed using a syringe and the precipitate was w hexanes (5 × 10 mL), then dried in vacuo to afford the titled compound as a film (22.5 mg, 0.0461 mmol, 66%).  ) and sodium ascorbate (5.55 mg, 0.0281 mmol, 0.4 equiv.) a mixture of t-BuOH and H 2 O (t-BuOH/H 2 O-1:1, 0.8 mL) was added.To this stirred mixture, phenylacetylene (23.3 µL, 0.211 mmol, 3.0 eq.) was then added, and the reaction mixture was stirred at 30 °C for 24 h.The reaction mixture was diluted with EtOAc (20 mL), washed with saturated aqueous NH 4 Cl solution (20 mL), dried over anhydrous MgSO 4 , filtered, and concentrated in vacuo.The obtained residue was then dissolved in a minimum amount of EtOAc, and to this vigorously stirred solution hexanes were added dropwise until precipitation started to occur.The mixture was stirred at rt overnight.The solvent was removed using a syringe and the precipitate was washed with hexanes (5 × 10 mL), then dried in vacuo to afford the titled compound as a thin, yellow film (22.5 mg, 0.0461 mmol, 66%).The General Procedure for the Pd/BIPHEPHOS reaction for the preparation of the cephamycin C-3 thiol derivatives 21a-h.Synthesis of the thiomethyl compound 21a is given as an example.
This compound was prepared following a procedure reported in the literature with some modifications [27].To a flame-dried flask Pd 2 (dba) 3 •CHCl 3 (10.7 mg, 0.0103 mmol, 10 mol%), BIPHEPHOS (17.0 mg, 0.0216 mmol, 21 mol%), and anhydrous MeCN (2 mL) were added under Ar at 50 • C. The resulting suspension was stirred at 50 • C for 30 min until it turned into a bright-yellow solution.The reaction flask was allowed to cool to rt and was then placed in a sonicator with the water temperature at 30-35 • C. Compound 20 (50.4 mg, 0.103 mmol) and 4-methoxybenzenethiol (25.4 µL, 0.207 mmol, 2.0 equiv.)were added to the reaction flask.The resulting mixture was sonicated at 30-35 • C until the completion of the reaction (10 h) was indicated using MS analysis.The reaction mixture was poured into EtOAc (10 mL) and extracted with NaHCO 3 (3 × 10 mL).The combined aqueous layer was acidified with conc.HCl to pH = 3 and extracted with EtOAc (3 × 10 mL).The combined EtOAc fraction was then washed with HCl (1.0 M-3 × 10 mL) and brine (20 mL), dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to give a yellow gum.This crude product was purified via semi-preparative RP-HPLC using a MeCN/H 2 O gradient mobile phase containing 0.01% TFA (MeCN/H 2 O-3:5-7:10, 15 min, injection volume = 200 µL) at a flow rate of 3.8 mL/min to give the titled compound as a pale-yellow oil (13.6 mg, 0.0240 mmol, 23%

Scheme 2 .
Scheme 2. Proposed commercially available starting materials (compounds 7 or 9) for the synthesis of cephamycin analogues 10 with variations at the C-7 amino group and the C-3 position.

Scheme 4 . 4 .
Scheme 4. Synthesis of diphenylmethyl ester 7 and PMB ester 8 and their α-bromoacetamide derivatives 17a and 17b, respectively.Compounds 17a and 17b are attractive intermediates for the synthesis of analogues related to the general structure 10 via the introduction of other substituents at the αacetamide and the C-3′ positions.To explore this potential, we have converted 17a and 17b to the carboxylic acid 18 via treatment with trifluoroacetic acid (TFA) (Scheme 5).Treatment of this acid with sodium azide in dimethyl formamide (DMF) at −5 °C gave the Scheme 4. Synthesis of diphenylmethyl ester 7 and PMB ester 8 and their α-bromoacetamide derivatives 17a and 17b, respectively.

Table 1 .
Attempts toward the synthesis of thiol 21a from triazole

20. Entry Ligand Ligand Equiv. Pd Equiv. Reaction Time Ratio of 20:21a a
a Determined using 1 H NMR analysis on the crude reaction mixture; b Reaction was performed under sonication; c 2.0 equiv. of 4-methoxybenzenethiol was used.